酿酒酵母苯丙氨酸代谢途径及分子调控机理研究

Saccharomyces cerevisiae is one of the most important industrial microorganisms, which is more safe and reliable than Escherichia coli in food fermentation. The color, taste and aroma of fermented food are closely related to microbial metabolite, especially the aromatic amino acids. S. cerevisiae has become a model species for understanding of metabolic regulation of higher organisms, as it is significant to annotate its metabolic network in “the era of post-genome”. Unfortunately, the accuracy for in silico prediction by the current two models (iFF708, iND750) is less than 85%; it is even more ambiguous in regulation genes and their functions of aromatic amino acids pathways. Our group has adequate experience in aromatic amino acid pathway engineering in E. coli and ethanol pathway engineering in S. cerevisiae. Based on prior researches, we plan to choose phenylalanine pathway of S. cerevisiae as object. Analyze and map the metabolic regulation network for phenylalanine pathway in S. cerevisiae from three aspects, the mechanism of feedback repression in synthetic pathway, the tuning points for enzyme synthesis and activity in degradation pathway, and transportation mode for amino acids. The results will contribute to illustrating the molecular regulation mechanism for phenylalanine metabolic pathway in S. cerevisiae, so as to provide theoretic basis for the artificial design of pathways for metabolites.

酿酒酵母是最重要的工业微生物之一，相对于大肠杆菌在食品发酵方面更加安全、稳定。发酵食品的色泽，口味及香气都与其代谢产物尤其是芳香族氨基酸密切相关。酿酒酵母业已成为理解高等生物代谢调控机制的主要模式生物，在后基因组时代，对其代谢网络的注释极为重要。然而，已有的酿酒酵母代谢网络模型(iFF708，iND750) in silico预测的准确率不足85%，无法精确、有效指导代谢途径的研究和改造；在芳香族氨基酸途径方面，部分调控基因及其调控机制仍十分模糊。本课题组结合酿酒酵母糖酵解途径和大肠杆菌氨基酸途径的代谢工程研究基础，以酿酒酵母苯丙氨酸代谢途径为研究对象，分别从合成代谢途径中的反馈阻遏机制，分解代谢途径中酶合成和酶活力的调节位点和氨基酸转运方式等方面开展研究。研究结果有助于阐明酿酒酵母苯丙氨酸代谢途径的分子调控机理，为酿酒酵母代谢产物的合理人工设计提供理论依据。

芳香族氨基酸在食品及医药领域的应用日益广泛。合成这一类化合物的途径是酿酒酵母细胞内极为重要的代谢途径，其不仅是细胞生存所必须，也为重要功能化合物的合成提供了前体。苯丙氨酸是研究最为深入的代表性芳香族氨基酸，然而其在酿酒酵母细胞内的代谢途径和调控机理尚不清楚。本项目首先开发和建立CRISPR/Cas9系统介导的高效基因编辑工具。以目标基因CDS上下游各45 bp碱基序列设计为同源臂供体，单基因敲除成功率超过90%。实现了对长度达26.5 kb的基因簇的敲除，成功率大于70%。利用同时搭载两个或三个sgRNA的表达载体，分别成功实现了两个不连续基因的一次敲入和多拷贝基因的敲入。为在基因组层面研究代谢途径奠定了基础。其次，在对苯丙氨酸合成途径的研究中发现，aro4编码的dahp同工酶活性受到酪氨酸的反馈抑制，229位置赖氨酸突变为亮氨酸可以成功解除酪氨酸的反馈抑制，且活性不受影响；aro7（催化分支酸转化合成预苯酸）141位置甘氨酸突变为丝氨酸后，可有效减轻酪氨酸的反馈抑制。aro10编码的苯丙酮酸脱羧酶发挥主要作用，在此基础上构建的重组菌细胞外L-苯丙氨酸的积累量达到5.16 mg/L。进一步的，将密码子优化后的YddG转运蛋白与EGFP进行融合后定位于细胞膜表达，胞外产物浓度提高至15.11 mg/L。最后，通过研究莽草酸合成途径的关键限速步骤，发现DAHP合酶和分支酸变位酶为代谢通路的瓶颈，并进一步阐述了PDC5对L-苯丙氨酸的合成和埃利希途径相关基因的调控。